Journal of J Appl Biotechnol Rep. 2019 Sep;6(3):96-100 Applied Biotechnology

doi 10.29252/JABR.06.03.03 Reports

Original Article

Investigating the Effect of Fennel and Cinnamon Combined Extract on Spermatogenesis and Testis Tissues in Busulfan Induced Infertile Rats Raziyeh Chegini1, Pouriya Soleimani1, Morteza Sadeghi2, Rana Mohammad Yosef1, Fariba Zafari3*

1Student Research Committee, Faculty of Medicine, Qazvin University of Medical Sciences, Qazvin, Iran 2Human Genetics Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran 3Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran

Corresponding Author: Fariba Zafari, Assistant Professor of Anatomical Sciences, Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran. Tel: +982833336003, Fax: +982833336006, Email: [email protected]

Received January 13, 2019; Accepted May 21, 2019; Online Published September 11, 2019

Abstract Introduction: Busulfan is one of the common cancer treatment drugs with infertility side effects. Fennel and cinnamon are two medicinal plants with fertility enhancement properties. The aim of this study was to investigate the effects of fennel and cinnamon on busulfan induced infertile rats. Materials and Methods: Forty male Wistar rats were divided into four groups including: sham group: healthy rats without intervention, control group: Busulfan treated rats, fennel group: busulfan and fennel extract treated, fennel and cinnamon group: busulfan, fennel and cinnamon extract treatment. Testicular tissues were sampled and the testicular physical parameters and spermatogenesis level were evaluated by H & E staining and optical microscopy imaging. Results: The biggest and the smallest testis lengths were observed in cinnamon + fennel and fennel groups respectively (P < 0.05). The highest and lowest sperm levels were observed in the cinnamon + fennel group and fennel group respectively (P < 0.001). The total average of reproductive cells was the most in the cinnamon + fennel group (208.88) and had the least level in the control group (81.2). Conclusions: The combined extract of fennel and cinnamon significantly protect the testicular tissues against infertility effect of busulfan. However, the fennel extract alone increased the effect of busulfan in rats. Keywords: Fennel, Cinnamon, Infertility, Spermatogenesis, Testis Citation: Chegini R, Soleimani P, Sadeghi M, Mohammad Yosef R, Zafari F. Investigating the effect of fennel and cinnamon combined extract on spermatogenesis and testis tissues in busulfan induced infertile rats. J Appl Biotechnol Rep. 2019;6(3):96-100. doi:10.29252/JABR.06.03.03.

Introduction (Foeniculum vulgare Mill) is an indigenous plant of Asia Spermatogenesis is a complex and multi-stage process. and Iran which is noticeable because of its health benefits Various factors such as stress, nutrition and certain drugs in traditional medicine.12 In recent research, fennel has been may affect this process and cause azoospermia or infertility. considered due to its antioxidant and anti-inflammatory One of the most common side effects of some chemotherapy properties.13,14 Also, a group of flavonoids called phytoestrogen drugs is men’s azoospermia.1-3 Busulfan is a leukemia exist in fennel seeds that have a competitive effect on binding treatment chemotherapy drug which is used before and to estrogen receptors. Actually, phytoestrogen maintains the after bone marrow transplantation. The administration of balance of unwanted reactions resulted from the changes in busulfan in male patients with malignant cancers can lead to feminine hormones before and after menopause in women.15 permanent or complete infertility.4,5 It seems that the disorder However, no studies have yet been done on the effects in spermatogenesis after busulfan treatment is due to the of fennel in spermatogenesis and infertility.16 Cinnamon busulfan alkylating effect on DNA, which mainly affects the (Cinnamomum zeylanicum) is a widely used alternative spermatogonia stem cells.6,7 So far, various studies have been medicine that is indigenous to India. It is an aromatic herbal conducted on the effects of busulfan on rat spermatogenesis plant that contains many antioxidant compounds such as during both fetal development8,9 and maturity stages.10,11 In all cinnamon aldehyde, terpenes, cinnamyl alcohol and safrole.17 of these studies, azoospermia and testis tissue damaging have Cinnamon is an extremely important plant in traditional been confirmed. Due to the high incidence of infertility in medicine and many properties, including fat reduction, patients undergoing chemotherapy, the use of new therapies antibacterial properties, antifungal properties and sexual has always been considered in different countries. Fennel enhancement properties have been reported to it.18,19 The

Copyright © 2019 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Effect of Fennel and Cinnamon Combined Extract on Spermatogenesis results from various studies confirm the fact that cinnamon measured and the specimens were then placed within a 48 extract has been effective in improving the function of the hour Buffin fixture. Then, the specimens were transferred reproductive system and increasing spermatogenesis in rats.20 to the microprocessor. After being overrun, the slices were Considering the increasing infertility in the males population stained with H & E staining. The ImageJ software was used and on the other hand, little research on the effect of fennel for the slides imaging. and cinnamon in the male reproductive system, in this study the synergistic effect of fennel and cinnamon extract and Statistical Analysis also the effect of fennel alone on the reproductive system of The results of the study were analyzed by SPSS and ANOVA. busulfan induced infertile rats has been investigated. The t test was used to evaluate the significant difference between groups and P ≤ 0.05 was considered as a significant Materials and Methods level for all calculations. Animal Preparation and Infertility Model For this study, 40 adult male Wistar rats (180-200 g) were Results prepared from the Razi Institute Center of Karaj, Iran. The Spermatogenesis Level of Studied Groups animals were administrated under standard laboratory Four weeks after treatments, the most spermatogonia number conditions according to the standard protocol at Qazvin was observed in the cinnamon + fennel group (73.19 ± 11.30) University of Medical Sciences Animal Breeding and and the lowest was observed in the fennel group (49.32 Development Center. The oligospermic model was performed ± 13.53). The difference between these two groups was by intravascular busulfan injection in two different dosages: significant (P < 0.05). The highest and lowest spermatocyte the first dosage of 25 mg/kg of busulfan and the second dosage numbers were observed in cinnamon + fennel (62.50 ± 13.70) of 15 mg/kg were administered 14 days later. and control group (13.67 ± 5.27) respectively. The highest and lowest spermatids numbers were observed in the cinnamon Animals Grouping and Treatment + fennel (73.19 ± 11.30) and control groups (15.7 ± 38.67) The rats were divided into 4 groups including sham group: respectively. Generally, the highest and lowest number of healthy rats without any intervention, control group: rats reproductive cells (spermatogonia, primary spermatocyte treated with busulfan in two doses of 25 mg/kg and 15 mg/ and spermatids) belonged to the fennel + cinnamon (208.88) kg with a 14 day interval, fennel group: Busulfan treated rats and control groups (81.2) (Table 1 and Figure 1). following with a fennel extract (100 mg/kg) intraperitoneal injection for a period of 5 days, and the fennel+cinnamon Testis Weight of the Studied Groups group: busulfan treated rats with a fennel (100 mg/kg) and In evaluation of the testis weight in the studied groups, the cinnamon (200 mg/kg) extract intraperitoneal injection for a testis weight in the fennel + cinnamon group was significantly period of 5 days. higher than the other groups. Among the groups, the highest testis weight (1.66 ± 0.17 g) belonged to the fennel + cinnamon Preparation of Fennel and Cinnamon Extract group and the lowest testis weight (1.22 ± 0.34 g) belonged Plants extraction was done by a pharmacologist at the Iranian to the fennel group, with a significant difference between the Research Institute of Pharmaceutical Sciences according to two groups (P < 0.05). The weight of the testis in the fennel the following protocol: 10 g of plant powder was added to 1 L group was even lower than the busulfan group (1.25± 0.89 g), of ethanol and extracted for 2 hours by ultrasonic apparatus. which indicates the negative effect of fennel on the testicular The extract was then placed in dark under laboratory (Table 2). conditions (25°C) for 24 hours. After filtration, the extract was concentrated by rotary evaporation (vacuum distillation) Testis Length and Width in Studied Groups at a temperature below 50°C. The extracts and fractions were In the evaluation of the size and length of the testis in the stored at -18°C until they were consumed. studied groups, the highest and lowest testis lengths was observed in fennel + cinnamon (2.32 ± 0.26 cm) and fennel Sampling and Assessments (1.56 ± 0.12 cm) groups respectively, and the difference According to the spermatogenesis period of rats which is between the two groups was statistically significant (P < 0.001). 4 weeks,21 the animal’s testis samples were prepared after The combined extract group had the best results regarding four weeks from the beginning of the treatment. For this the size of the testis compared to the control group. The testis purpose, after the anesthetic, the testis were removed and length in the fennel group (1.56 ± 0.12) was even lower than the parameters of weight, length and width of them were the busulfan treated group (2.17 ± 0.18). In the evaluation

Table 1. Reproductive Cells Count in the Study Groups on the 14th Day

Spermatogonia Primary Spermatid Spermatid P Value Sham 69.17 ± 12.28 76.50 ± 16.67 85 ± 13.9 - Control 51.83 ± 23.37 13.67 ± 5.27 15.7 ± 38.67 - Fennel 49.32 ± 13.53 57.20 ± 12.95 65.02 ± 18.8 - Fennel + Cinnamon 73.19 ± 11.30 62.50 ± 13.70 73.19 ± 11.30 0.001

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120 Spermatid Primary spermatocyte Spermatogoni

100

80

60

Number of cells 40

20

0 Sham Control Fennel Fennel+Cinnamon

Groups

Figure 1. Comparison of Reproductive Cells in the Studied Groups. The average number of spermatogonia cells in the fennel + cinnamon group was increased, but this increase was significant in comparison with the fennel group (P <0.05). The number of primary spermatocytes in the control group (busulfan) was significantly lower than the other groups (P < 0.001). The number of spermatids in the combined group increased compared to the other groups, which was significantly higher than the fennel group and control Was there (P < 0.001).

of the testis width, the biggest and smallest testis width were This is while the use of fennel extract alone exacerbated the observed in the fennel+cinnamon (1.41± 0.48 cm) and fennel side effects of busulfan and led to the loss of testis tissue and (1.11± 0.16 cm) groups respectively (Figure 2 and Table 2). reduction of spermatogenesis. According to the results in new research, cinnamon could Discussion increase sperm quality parameters such as population, In this study, the effect of fennel and cinnamon extract was viability and motility. Herbal antioxidants eliminate and investigated on busulfan-induced infertile rats. According to suppress reactive oxygen species (ROS) formation, and the the findings of the present study, fennel and cinnamon mixed reduction of ROS is a crucial factor in the production of sperm extracts repair the testis tissue and significantly increase cells and optimization of the fertility rate.22,23 More research the spermatogenesis level in busulfan-induced infertile rats. seems to be necessary to confirm the pharmacological and toxicological effects of this medical herb on body tissues.24

Table 2. Testicular Physical Parameters of the Studied Groups on the 14th Antol is a substance that acts like estrogen. It has been Day reported that its consumption in men decreases testosterone 25 Weigh (g) Length (cm) Width (cm) levels and depletes sexual power. These observations Sham 1.47±0.16 2.50±0.25 1.48±0.38 are consistent with the findings of Mansouri et al who have reported that fennel leads to a reduction of blood Control 1.25±0.89 2.17±0.18 1.36±0.19 testosterone and spermatogenesis in rats.26 In a study on Fennel 1.22±0.34 1.56±0.12 1.11±0.16 the effect of intraperitoneal injection of fennel extract on Fennel + Cinnamon 1.66±0.17 2.32± 0.26 1.41±0.48 rat spermatogenesis, Rezaei Ahvanouei et al reported that the fennel extract significantly reduced the number of spermatid.27 In the present study, the most reduction in testis size was observed in the simultaneous use of fennel and busulfan, which confirms previous findings. The study of the effects of the aqueous extract of fennel seed by Hosseini Ahar et al indicated that busulfan injection reduced testis weight and increased serum levels of MDA.28 Ghasemi et al reported that busulfan reduced the production of spermatogonia cells in mice.29 This finding suggest that fennel exacerbates the infertility effects of busulfan. Antol and fennel are to important compounds present in the fennel extract.30,31 Contrary to the results of this study, Mansouri and colleagues reported that histological and morphometrical studies of testis tissue indicated a reduction in sperm cell count in the experimental groups compared to the control group. They suggested that hydroalcoholic extract of fennel seed had inhibitory effect on spermatogenesis and fertility.26 One of the Figure 2. H & E Staining Image of Seminal Tube Sections of the Studied Groups. main reasons for reduction in the cell count could be due to A: Control group (Busulfan), B: Sham group (B1: spermatogonia, B2: primary the existing compounds in the herb extract that inhibited cell spermatocyte, B3: spermatid), C: Fennel group, D: Fennel + Cinnamon group. division, particularly flavonoids. According to the findings of

98 J Appl Biotechnol Rep, Volume 6, Issue 3, 2019 http://www.biotechrep.ir The Effect of Fennel and Cinnamon Combined Extract on Spermatogenesis recent studies, flavonoids could restrain DNA replication and transplantation in patients with refractory lymphoid malignancies. inhibit cell division, as well as stimulate apoptosis by affecting Biol Blood Marrow Transplant. 2012;18(11):1677-1686. 32 doi:10.1016/j.bbmt.2012.05.011. cell DNA. 7. Choi YJ, Ok DW, Kwon DN, et al. Murine male germ cell Garawani et al reported that the consumption of fennel apoptosis induced by busulfan treatment correlates with loss of oil in etoposide defective rats caused reduction of sperm c-kit-expression in a Fas/FasL-and P53-independent manner. FEBS defects and increased mature sperm production, which was Lett. 2004;575(1-3):41-51. due to the anti-oxidant properties of the fennel extract.33 The 8. Boujrad N, Hochereau-de Reviers MT, Kamtchouing P, Perreau C, findings of Garawani et al’s study about the effect of fennel Carreau S. Evolution of somatic and germ cell populations after busulfan treatment in utero or neonatal cryptorchidism in the rat. on spermatogenesis are contradictory to the findings of Andrologia. 1995;27(4):223-228. doi:10.1111/j.1439-0272.1995. this study. In evaluation of fennel and cinnamon combined tb01097.x. extract, a significant increase has been observed in the testis 9. Jansz GF, Pomerantz DK. The effect of prenatal treatment with reproductive cells especially in the spermatogonia cells. In busulfan on in vitro androgen production by testes from rats of related studies, Khaki et al reported that consuming cinnamon various ages. Can J Physiol Pharmacol. 1985;63(9):1155-1158. doi:10.1139/y85-189. alone or mixed with fennel will significantly increase the 10. Vecino P, Uranga JA, Arechaga J. Suppression of spermatogenesis for 34 number, motility and survival of sperms. cell transplantation in adult mice. Protoplasma. 2001;217(4):191- 198. doi:10.1007/BF01283400. Conclusions 11. Haddad S, Carvalho TL, Anselmo-Franci JA, Petenusci SO, Favaretto In conclusion, the findings of this study suggest that the AL. Ultrasound stimulation of rat testes damaged by busulfan. hydro-alcoholic extract of fennel and cinnamon could repair Ultrasound Med Biol. 1997;23(9):1421-1425. doi:10.1016/s0301- 5629(97)00185-3. the damaged testis tissues and improve the spermatogenesis 12. Albert-Puleo M. Fennel and anise as estrogenic agents. J process in busulfan induced rats and it seems that these plant Ethnopharmacol. 1980;2(4):337-344. doi:10.1016/s0378- compounds can be used to treat chemotherapy-induced 8741(80)81015-4. infertility in the future. 13. Carr DW, Usselman MC, Acott TS. Effects of pH, lactate, and viscoelastic drag on sperm motility: a species comparison. Biol Authors’ Contributions Reprod. 1985;33(3):588-595. doi:10.1095/biolreprod33.3.588. FZ and RC designed the study and performed the experiments. 14. Shah AH, Qureshi S, Ageel AM. Toxicity studies in mice of MS analyzed the data and wrote the paper.PS and RY performed ethanol extracts of Foeniculum vulgare fruit and Ruta chalepensis experiments and collected samples. FZ analyzed the data and critically aerial parts. J Ethnopharmacol. 1991;34(2-3):167-172. reviewed the manuscript. doi:10.1016/0378-8741(91)90034-b. 15. Malini T, Vanithakumari G, Megala N, Anusya S, Devi K, Elango Conflict of Interest Disclosures V. Effect of Foeniculum vulgare Mill. seed extract on the genital The authors declare they have no conflicts of interest. organs of male and female rats. Indian J Physiol Pharmacol. 1985;29(1):21-26. Funding 16. Kooti W, Moradi M, Ali-Akbari S, Sharafi-Ahvazi N, Asadi-Samani This project was funded by the financial support of the Student Research M, Ashtary-Larky D. Therapeutic and pharmacological potential Committee, School of Medicine, Qazvin University of Medical Sciences. of Foeniculum vulgare Mill: a review. J Herbmed Pharmacol. 2015;4(1):1-9. Acknowledgments 17. Anderson RA, Broadhurst CL, Polansky MM, et al. Isolation The authors would like to sincerely thank the Student Research and characterization of polyphenol type-A polymers from Committee of Qazvin University of Medical sciences for providing the cinnamon with insulin-like biological activity. J Agric Food Chem. needed facilities of this study. 2004;52(1):65-70. doi:10.1021/jf034916b. 18. Khan A, Safdar M, Ali Khan MM, Khattak KN, Anderson RA. References Cinnamon improves glucose and lipids of people with type 2 1. Nudell DM, Monoski MM, Lipshultz LI. Common medications diabetes. Diabetes Care. 2003;26(12):3215-3218. doi:10.2337/ and drugs: how they affect male fertility. Urol Clin North Am. diacare.26.12.3215. 2002;29(4):965-973. doi:10.1016/S0094-0143(02)00079-4. 19. Nir Y, Potasman I, Stermer E, Tabak M, Neeman I. Controlled trial of 2. Puscheck E, Philip PA, Jeyendran RS. Male fertility preservation the effect of cinnamon extract on Helicobacter pylori. Helicobacter. and cancer treatment. Cancer Treat Rev. 2004;30(2):173-180. 2000;5(2):94-97. doi:10.1046/j.1523-5378.2000.00014.x. doi:10.1016/j.ctrv.2003.07.005. 20. Kamath JV, Rana AC, Chowdhury AR. Pro-healing effect of 3. Shetty G, Meistrich ML. Hormonal approaches to preservation and Cinnamomum zeylanicum bark. Phytother Res. 2003;17(8):970- restoration of male fertility after cancer treatment. J Natl Cancer 972. doi:10.1002/ptr.1293. Inst Monogr. 2005(34):36-39. doi:10.1093/jncimonographs/ 21. Kierszenbaum AL. Histology & cell biology: an introduction to lgi002. pathology. 2nd ed. Saunders; 2006. 4. Anjamrooz SH, Movahedin M, Mowla SJ, Bairanvand SP. 22. Bordbar H, Esmaeilpour T, Dehghani F, Panjehshahin MR. Assessment of morphological and functional changes in the mouse Stereological study of the effect of ginger’s alcoholic extract on testis and epididymal sperms following busulfan treatment. Iran the testis in busulfan-induced infertility in rats. Iran J Reprod Med. Biomed J. 2007;11(1):15-22. 2013;11(6):467-472. 5. Temitope AA, Tolulope OA, Funlola CT, Khaleed TB, Effiong 23. Henkel R. The impact of oxidants on sperm function. Andrologia. UA, Adetola AA. Protective effect of Tahitian Noni juice on 2005;37(6):205-206. doi:10.1111/j.1439-0272.2005.00699.x. the reproductive functions of Male Wistar Rats traeted with 24. Khaki A, Fathiazad F, Nouri M, et al. Beneficial effects of quercetin cyclophosphamide. Iranian Journal of Pharmacology and on sperm parameters in streptozotocin-induced diabetic male rats. Therapeutics. 2011;10(1):39-43. Phytother Res. 2010;24(9):1285-1291. doi:10.1002/ptr.3100. 6. Nieto Y, Thall P, Valdez B, et al. High-dose infusional gemcitabine 25. Mokhtari M, Sharifi E, Moghadamnia D. Effect of alcoholic extract combined with busulfan and melphalan with autologous stem-cell of phoenix dactylifera spathe on histological change in testis and http://www.biotechrep.ir J Appl Biotechnol Rep, Volume 6, Issue 3, 2019 99 Chegini et al

concentrations of LH, FSH and testosterone in male rat. Iran J Basic 30. Khouri NA, El-Akawi Z. Antiandrogenic activity of Ruta graveolens Med Sci. 2007;9(4):265-271. L in male Albino rats with emphasis on sexual and aggressive 26. Mansouri E, Asadi-Samani M, Kooti W, et al. Anti-fertility effect of behavior. Neuro Endocrinol Lett. 2005;26(6):823-829. hydro-alcoholic extract of fennel (Foeniculum vulgare Mill) seed 31. Namavar Jahromi B, Tartifizadeh A, Khabnadideh S. Comparison in male Wistar rats. J Vet Res. 2016;60(3):357-363. doi:10.1515/ of fennel and mefenamic acid for the treatment of primary jvetres-2016-0052. dysmenorrhea. Int J Gynaecol Obstet. 2003;80(2):153-157. 27. Rezaei Ahvanouei T, Shiravi A, Keramati K. Stady of IP injection of doi:10.1016/s0020-7292(02)00372-7. “FoeniculumVulgare mill” seed aqua extract on Spermatogenesis 32. Moskot M, Jakobkiewicz-Banecka J, Smolinska E, et al. Effects of and Histologic changes of in male Wistar rats. Journal of Animal flavonoids on expression of genes involved in cell cycle regulation Biology. 2009;2(1):19-26. and DNA replication in human fibroblasts. Mol Cell Biochem. 28. Hosseini Ahar N, Khaki A, Akbari G, Ghaffari Novin M. The effect of 2015;407(1):97-109. doi:10.1007/s11010-015-2458-3. busulfan on body weight, testis weight and MDA enzymes in male 33. El-Garawani I, El-Nabi SH, El-Ghandour E. The protective effect rats. International Journal of Women’s Health and Reproduction of (Foeniculum vulgare) oil on etoposide-induced genotoxicity on Sciences. 2014;2(5):316-319. doi:10.15296/ijwhr.2014.52. male albino rats. Eur J Pharm Med Res. 2017;4(7):180-194. 29. Mohammadghasemi F, Soleimanirad J, Ghanbari AA. An 34. Khaki A. Effect of Cinnamomum zeylanicumon on Ultrastructural Study on the Apoptotic Features of Spermatogenic Spermatogenesis. Iran Red Crescent Med J. 2015;17(2):e18668. Cells following Busulfan Treatment in Adult Mice. J Reprod Infertil. doi:10.5812/ircmj.18668. 2008;8(4):319-329. [Persian].

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